Glucocorticoids have a broad array of life-sustaining functions and play an important role in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Thus, changes of tissue responsiveness to glucocorticoids may develop into pathologic states and influence their course. We investigated pathophysiologic mechanism of one of such conditions, familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We examined molecular defects of known pathologic mutants GRR477H and GRG679S. GRR477H completely lost its transcriptional activity due to its inability to bind the glucocorticoid response elements (GREs), while GR679S demonstrated reduced transcriptional activity with attenuated ligand-binding activity. We recently found two new heterozygotic mutations that replace aspartic acid by histidine at amino acid 401 and phenylalanine by leucine at amino acid 737 (GRD401H and F737L). In preliminary experiments, GRD401H demonstrated 2-3 times stronger transcriptional activity than the wild type receptor, while GRF737L produced a right-shifted dexamethasone-titration curve of transcriptional activity. We will examine their molecular defects and clarify impact of the mutations found to patients? phenotypes. To examine biologic defects of pathologic GR mutant receptors in living cells, we examined their motility inside the nucleus by using the fluorescence recovery after photobleaching (FRAP) method. Motility of the ligand-bound wild type GR is regulated inside the nucleus through its multiple interactions with the chromatin-associated molecules, transcriptional intermediate proteins, target DNAs and the ubiquitine/proteasomal pathway. We found that all of the examined mutant receptors demonstrated increased motility, which was correlated with their ligand-dependent transcriptional activity. Thus, pathologic mutant GRs possess dynamic motility defects in the nucleus, possibly caused by their inability to properly interact with key partner nuclear molecules necessary for full activation of glucocorticoid-responsive genes. To find intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings using several GR domains as baits. We found in the screening using the N-terminal domain of GR as bait that the transforming growth factor (TGF) beta/bone morphogenetic protein-downstream Smad6 specifically interacted with GR. Smad6 suppressed GR transcriptional activity both at cellular and animal levels via attracting histone deacetylases and antagonizing histone acetylation induced by p160 type histone acetyltransferase coactivators. It appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anti-catabolic and pro-wound healing properties of the TGF beta family of proteins. In another yeast two-hybrid screening using the GR DNA-binding domain, we found that SET/TAF-1beta and gas5 interacted with this portion of the GR. The former molecule is known as a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex that binds lysine residues of histones and protects them from acetylation by histone acetyltransferases. We found that SET/TAF-1beta acts as a negative regulator of GR transcriptional activity and ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through the DBD. In contrast to Set/TAF-I beta, the Set-Can fusion protein constitutively bound GREs and suppressed GR-induced transcriptional activity/histone acetylation regardless of ligand availability. Thus, Set/TAF-I beta acts as a dynamic regulator of GR-induced transcriptional activity, suppressing baseline transcription by preventing histone acetylation, while facilitating a switch from INHAT-corepressor to HAT-coactivator complexes on GR-bound promoters in response to ligand. Pathologic fusion of Set to Can might disrupt this physiologic switch, causing glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation. The other interacting molecule gas5 that was found in the same screening is non protein-coding RNA. It is highly accumulated in growth-arrested cells, but its physiologic roles are not known. We found that gas5 bound GR at its DNA-binding domain, prevented its association with GREs and suppressed its transcriptional activity. Serum starvation-induced gas5 suppressed glucocorticoid-mediated cellular inhibitor of apoptosis 2 mRNA expression and prevented apoptosis of growth-arrested cells. In addition to GR, gas5 suppressed the transcriptional activities of the progesterone and estrogen receptors, but not of other transcription factors. Thus, gas5 is a growth arrest-related co-repressor of steroid receptors in resting cells, restricting the expression of steroid-responsive genes, whose expression might be nonadaptive in growth factor-deprived cells. Glucocorticoids play an essential role in the homeostasis of the central nervous system and influence diverse functions of neuronal cells. In a cyto-trap yeast two-hybrid screening using the GR ligand-binding domain, we found that cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of the nervous system, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with this domain of the GR through its activators p35/p25. CDK5 phosphorylated GR at serines 203 and 211 located in its N-terminal domain and suppressed the transcriptional activity of this receptor on a glucocorticoid-responsive promoter by attenuating attraction of transcriptional cofactors to DNA. In microarray analyses using rat cortical neuronal cells, the CDK5 inhibitor roscovitine differentially regulated the transcriptional activity of the GR on more than 90 percent of the endogenous glucocorticoid-responsive genes tested. Thus, CDK5 exerts some of its biologic activities in neuronal cells through the GR, dynamically modulating GR transcriptional activity in a target promoter-dependent fashion. Brx, a Rho type guanine nucleotide exchange factor, activates Rho family small G proteins by converting them from inactive GDP-bound to active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We found that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to GRE-bound GR. Brx mediated lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. LPA is produced from activated platelets in patients with dysmetabolic syndrome who may have glucocorticoid hypersensitivity. Thus, Brx may contribute to theincreased glucocorticoid sensitivity seen in these patients, mediating LPA-induced signal to GR. Circulating glucocorticoid concentrations fluctuate in a circadian pattern coordinated with the animal?s rest-activity cycle, such that peak concentrations are reached around the time of onset of the active period and lowest concentrations in association with inactivity. The circadian clock, which consists of the basic helix-loop-helix transcription factors CLOCK and its hetero-dimer partner BMAL1, controls circadian rhythms both in the central nervous system and peripheral tissues. In preliminary experiments, we found that the CLOCK/BMAL1 expression potentiated GR-induced transcriptional activity by directly acetylating the GR. Thus, it appears that the circadian rhythm regulates glucocorticoid action at the level of target tissues through the interaction between GR and CLOCK/BMAL1.